The adenovirus L4-22K protein is multifunctional and critical for different aspects of viral infection. Packaging of the viral genome into an empty capsid absolutely requires the L4-22K protein to bind to viral DNA packaging sequences in cooperation with other viral packaging proteins. Additionally, the L4-22K protein is important for the temporal switch from the early-to-late phase of infection by regulating both early and late gene expression. Our results demonstrate that these two functions of L4-22K may be uncoupled. L4-22K functions in the temporal control of viral gene expression by activating transcription from the major late promoter, and more importantly, by regulating the accumulation of specific adenovirus late transcripts. Our results reveal that the L4-22K protein controls viral gene expression at the post-transcriptional level and regulates alternative pre-mRNA splicing. Specifically, L4-22K promotes the splicing of its own transcript to generate a new transcript that encodes the L4-33K protein. This L4-22K function is conserved among evolutionarily diverse human adenoviruses. We propose a feedback loop, wherein L4-22K regulates accumulation of different L4 transcripts and gene products. As L4-22K protein levels increase, L4-33K pre-mRNA splicing is stimulated. Since L4-33K itself regulates Ad late mRNA splicing and is required for the appropriate accumulation of Ad late transcripts, this finely tunes the temporal switch of gene expression patterns during the late phase of infection. We hypothesize that the L4-22K protein functions via specific cis-acting sequences in viral pre-mRNAs and via specific interaction with a cellular splicing factor(s). In this application, we propose to use genetic and biochemical approaches to probe the mechanism by which L4-22K regulates pre-mRNA splicing. Alternative pre-mRNA splicing is widely utilized by DNA viruses and complex retroviruses yet little is known about underlying mechanisms of regulation. Further, there is increasing recognition of the role of aberrant pre-mRNA splicing underlying many human diseases and this forms a basis for therapeutic intervention. The results of the proposed research will provide novel information regarding the regulation of alternative pre-mRNA splicing in eukaryotic cells and in the context of viral infection.